A major recent advancement for the field of pain research is the recognition of immune system dysregulation as a contributor to the most serious adverse outcomes from injury. Both autoinflammation involving mediators of inflammation and autoimmunity involving autoantibodies have been identified in patients with chronic pain and disability after limb trauma. Taking advantage of these advancements, the overarching goal of the proposed research is to identify novel approaches to controlling immune system activation after limb injury thereby increasing the rate and improving the quality of recovery while reducing chronic pain, functional loss and neuropsychiatric consequences such as anxiety and cognitive decline. Building on evidence from the fields of immunology, pain and rehabilitation, this project will focus on the autonomic regulation of the immune system after injury. The proposed experiments involve a well-validated rodent tibial fracture model of limb injury as well as samples obtained from patients with chronic limb pain after traumatic events. Our central hypothesis is that interruption of a sympathetically-initiated immunological cascade will greatly enhance the rate and quality of recovery by reducing IL-6 production, autoantibody formation and activation of the complement cascade. The project is divided into three aims. In the first aim we will determine whether modulation of autonomic outflow after tibial fracture reduces immune activation and improves key dimensions of recovery. We hypothesize that post-traumatic activation of the sympathetic nervous system sets an immunological pathway in motion including IL-6 expression, IgM autoantibody production and complement activation ultimately leading to chronic pain, functional decline, anxiety and cognitive decline. Activation of the parasympathetic nervous system may have opposite effects. Using neuroablative and pharmacological approaches, we will test this hypothesis using a panel of outcome measures designed to address multiple dimensions of recovery. In the project?s second aim we focus strongly on the roles of IL-6 signaling after limb injury as a mediator supporting IgM autoantibody production. We hypothesize that blocking the sympathetically-enhanced production of IL-6 after limb injury will reduce autoantigen expression in the injured limbs, regional lymph node hypertrophy, B-cell differentiation and IgM autoantibody production. We will use genetic tools as well as clinically available biologic and small molecule anti-IL-6 agents to define the role of this cytokine in supporting the long-term adverse outcomes of limb injury. Finally, the project?s third aim is to determine whether injury-related autoantibodies support adverse outcomes via peripheral and central complement system activation. We hypothesize that IgM autoantibodies produced by limb injured mice and, translationally, limb injured patients support complement cascade activation leading to the generation of pain-promoting C5a and neuron injuring C5b-9 membrane attack complexes. We predict that genetic complement component deletion and pharmacological C5a receptor blockade will accelerate recovery after limb fracture. In addition, we predict that the local injection of injured mouse and human IgM will cause pain sensitization and functional loss in the involved limbs. Biochemical and immunohistochemical studies will identify local complement activation and fiber loss. Chronic pain syndromes after injuries and surgery including CRPS have substantial pain, functional, neuropsychiatric and financial consequences. Available treatments are poorly effective. At the conclusion of these experiments we will be in position to design clinical protocols evaluating the impact of sympatholysis, immunomodulation and anti-complement therapeutics on recovery from injury using clinically available tools and ones currently in late stages of development.